Extracorporeal affinity adsorption methods for the treatment of atherosclerosis, cancer, degenerative and autoimmune diseases

ABSTRACT

Extracorporeal affinity adsorption treatments which are aimed at the substantial removal of two or more compounds that are etiological in the pathogenesis of diseases in man provide effective therapeutic intervention means for these diseases. The methods are particularly suitable for the treatment of atherosclerosis, cancer, degenerative and autoimmune diseases. Extracorporeal chelation and immunotherapy for atherosclerosis, extracorporeal chelation treatment with on-line regeneration or replacement of chelant, extracorporeal immunotherapy with antibody fragments, and extracorporeal immunoadsorption utilizing antibodies bound to Protein A are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a division of pending application Ser. No. 09/007,599,filed Jan. 15, 1998, which is a division of Ser. No. 08/097,378, filedJul. 23, 1993, now U.S. Pat. No. 5,753,227, the disclosure of which ishereby incorporated by reference herein.

BACKGROUND OF THE INVENTION

[0002] Atherosclerosis and cancer are the two major causes of morbidityand mortality in western societies. While there has been significantadvance in the treatment of atherosclerosis there is still a great needfor more effective treatment interventions.

[0003] The main mechanism by which atherosclerosis leads to morbidityand mortality is by narrowing the lumen of arteries and reducing theblood supply to the heart, brain and other vital organs. The factorsassociated with atherosclerosis include: High levels of cholesterol,triglycerides, low density lipoproteins (LDL) and low levels of highdensity lipoproteins (HDL).

[0004] Other factors are heredity, cigarette smoking, obesity, highblood pressure, reduced physical activity, high fat diets, and a highoxidation activity associated with the production of free radicals,leading to the oxidation of LDL, which accelerates the development ofatherosclerotic lesions. Thus, J. Regnstrom et al., Lancet, Vol. 339,No. 8803, May 16, 1992, pp. 1183-86, reported that the susceptibility ofLDL to in vitro oxidation in the presence of copper, which acts as acatalyst in the oxidation process, was correlated with the severity oftheir coronary artery sclerosis. J. T. Salonen et al, Lancet, Vol. 339,No. 8798, Apr. 11, 1992, pp. 883-87, found that the level ofautoantibodies to oxidized LDL predicted the progression ofatherosclerosis of the carotid artery (the artery that supplies blood tothe brain). One likely mechanism in development of atheroscleroticlesions via the oxidation of LDL is the induction of an autoimmuneprocess leading to the production of antibodies specific to oxidized LDLand propagation of the atherosclerotic lesion by the autoantibodybinding to oxidized LDL. J. Regnstrom et al., supra, and T. Kita et al.,Proceedings of the National Academy of Sciences USA, Vol. 84, 1987, pp.5928-31. J. T. Salonen et al., Circulation, Vol. 86(3), September 1992,pp. 803-11 reported an association between the risk of heart attack andthe level of iron in the blood, with the risk being particularly highwhen plasma levels of both iron and LDL were elevated.

[0005] A significant reduction in blood levels of LDL and cholesterol bydiet and lipid reducing drugs was found to result in regression ofatherosclerosis. G. Brown et al., New England Journal of Medicine, Vol.323(19), Nov. 8, 1990, pp. 1289-1298. Oral lipid lowering drugs, such asLovastatin, MSD (Mevacor®, Merck), are risky and may cause liver damage.Their efficacy is relatively limited, even when they are taken inassociation with a strict diet. Lowering of LDL by extracorporealtreatment of blood, M. Strahilevitz, U.S. Pat. Nos. 4,375,414 and4,813,924, and M. Strahilevitz, Atherosclerosis, Vol. 26, 1977, pp.373-77, is significantly more effective in reducing blood cholesteroland LDL levels. H. Borberg et al., Journal of Clinical Apheresis, Vol.4, 1988, pp. 59-65; R. L. Wingard et al., American Journal of KidneyDiseases, Vol. 18(5), 1991, pp. 559-65; V. Hombach et al., Dtsch Med.Wschr, Vol. 111(45), 1986, pp. 1709-15. LDL and cholesterol can beremoved by affinity adsorption, utilizing as the adsorbent antibodies toLDL or other specific chemical adsorbents, such as dextran sulphate (M.Odaka et al., International Journal of Artificial Organs, Vol. 9, 1986,pp. 343-48) or heparin (D. J. Lupien et al., Pediatric Res., Vol. 14,1980, pp. 113-17). LDL removal can also be achieved by heparinprecipitation (D. Seidel et al., Journal of Clinical Apheresis, Vol. 4,1988, pp. 78-81), and by double filtration plasmapheresis (S. Yokoyamaet al., Arteriosclerosis, Vol. 5, November/December 1985, pp. 613-22) aswell as by plasma exchange (G. R. Thompson, Lancet, 1981 I, pp.1246-48).

[0006] The oral administration of vitamin E is associated with lowerrisk of coronary heart disease in men (E. B. Rimm et al., New EnglandJournal of Medicine, Vol. 328(20), May 20, 1993, pp. 1450-56) and inmiddle aged women (M. J. Stampfer et al., New England Journal ofMedicine, Vol. 328(20), May 20, 1993, pp. 1487-89). The mechanism ofthis protective effect is based on the antioxidant property of vitaminE, which inhibits the oxidation of LDL, thus exerting a protectiveeffect from the development of atherosclerosis. The oxidation of LDL iscatalyzed by heavy metals such as iron and copper. The removal of themetals by intravenous administration of chelating agents was reported tobe effective in atherosclerotic vascular disease. E. Olszewer and J. P.Carter, Medical Hypotheses, Vol. 27(1), September 1988, pp. 41-49, andE. Cranton, “Bypassing Bypass,” Hampton Road Publishers, Norfolk, Va.,1992. Others did not confirm these reports. S. R. Wirebaugh and D. R.Gerates, DICP, Vol. 24(1), January 1990, pp. 22-24.

[0007] The apparent minimal effect, or lack of effect, of intravenouschelation in the treatment of atherosclerosis can be overcome byextracorporeal chelation which significantly increases chelationefficacy and reduces significantly its toxicity. M. Strahilevitz,Lancet, Vol. 340, Jul. 25, 1992, p. 235.

[0008] Extracorporeal chelation with desferoxamine was highly effectiveand safe in reducing blood iron in the treatment of hemochromatosis, adisease caused by the accumulation of excess iron in the blood and bodystores. J. L. Held et al., Journal of American Academy ofDermatologists, Vol. 28, 1993, pp. 253-54. Ambrus and Horvath in U.S.Pat. No. 4,612,122 also describe a specific column configuration thatcan be used for extracorporeal chelation. In this column the chelatingagent is physically immobilized in the spongy outer part of ananisotropic (asymmetrical) membrane.

[0009] Chelating agents can also be utilized with the extracorporealaffinity adsorption devices of Strahilevitz, U.S. Pat. No. 4,375,414.

[0010] Coronary bypass surgery is effective in reducing symptomatology,but its effect on mortality is limited. J. H. O'Keefe, Jr. and B. D.McCallister, Editorial, Mayo Clinic Proceedings, Vol. 67, 1992, pp.389-91, R. D. Simari et al., Mayo Clinic Proceedings, Vol. 67, April1992, pp. 317-22.

[0011] Bypass surgery has no curative effect on the atheroscleroticdisease process. The problem of post surgery atherosclerosis progressionand the development of coronary or graft restenosis are major problemsassociated with bypass surgery. The need for effective means forreducing progression and inducing regression of atherosclerosis inpatients following bypass surgery is well recognized, as is the need tofurther develop effective nonsurgical treatments that would replacebypass surgery in a significant proportion of patients that arecurrently being treated with bypass surgery, because of the lack ofalternative effective medical treatment.

[0012] This is particularly relevant for candidates for bypass coronarysurgery with moderately severe coronary occlusion that may not exhibitsignificant fibrotic changes in the atherosclerotic coronary lesions.Similar limitations to those of bypass surgery apply to percutaneoustransluminal coronary angioplasty. Simari et al., supra. In thisprocedure, an inflatable balloon is inserted into the coronary occlusionsite. As with bypass surgery, this procedure also has no effect on theatherosclerotic disease process, thus restenosis is a significantproblem. While the risks associated with angioplasty are lower than withbypass surgery, this is also an invasive procedure associated withmorbidity and mortality risks.

[0013] While current medical treatments, particularly when combinationsof conventional treatments are utilized, have significant effect inreducing progression and in inducing regression of the atheroscleroticprocess (Brown et al., supra), there is a need to have more effectivetreatment methods, particularly for those who can not be treated withoral lipid lowering drugs because of liver toxicity, who are unable tomaintain a strict diet, or who fail to improve with conventionaltreatment, including oral lipid lowering drugs and diet.

[0014] The utilization of extracorporeal affinity adsorption of LDL(Strahilevitz, supra) can lead to marked reduction in LDL level, thus tosignificant regression of atherosclerotic coronary lesions. Hombach etal., supra. However, the effect of affinity adsorption of LDL andcholesterol, while aimed at a major factor in atherosclerosis,hyperlipidemia, is selectively targeted on this factor. Even when (asusually is the case) the affinity LDL adsorption is utilized with othermeasures (diet, exercise etc.) the quantitative impact of theseconventional treatment methods may not be sufficient. The availabilityof non-surgical methods that will have a significantly largerquantitative effect on additional factors that are involved in theetiology and pathogenesis of atherosclerosis is of great importance, inorder to optimize the non-surgical and post-surgical treatment ofatherosclerosis.

SUMMARY OF THE INVENTION

[0015] One of the objects of the present invention is to provideeffective non-surgical treatments of atherosclerosis.

[0016] Another object is to provide improvements in extracorporealtreatment methods for atherosclerosis and other diseases.

[0017] Another object is to provide improved specific affinity devices,particularly immunoadsorption devices, and methods.

[0018] Other objects will become apparent to those skilled in the art inlight of the following description.

[0019] In accordance with one aspect of the present invention, methodsand devices for treating atherosclerosis and other conditions areprovided that are based on the utilization of specific affinityadsorption of several of the biological molecules that are etiologicalin the pathogenesis of the condition. The affinity adsorbents utilizedin accordance with the present invention are both immunoadsorbents andnon-immune-based specific affinity chemical adsorbents.

[0020] In some applications of extracorporeal combined treatment, one orboth of the extracorporeal methods may be based on other principles thanadsorption, for example use of extracorporeal double filtration for theremoval of LDL. S. Yokoyama et al., supra.

[0021] The adsorbents are incorporated in an extracorporeal treatmentdevice. The methods of the present invention will be usually utilized inconjunction with conventional treatment methods, both medical and, whenindicated, surgical methods.

[0022] The novel treatment methods that are the subject of the presentinvention are based on and are specific improvements of extracorporealaffinity adsorption and extracorporeal affinity dialysis which aredisclosed in Strahilevitz U.S. Pat. Nos. 4,375,414 and 4,813,924 andBritish provisional patent application No. 16001, May 20, 1971, andwhich are incorporated herein by reference.

[0023] It is one of the objects of the present invention to provideadditional specific improvements and embodiments to further increase theeffectiveness and utility of extracorporeal affinity adsorptiontreatment of atherosclerosis.

[0024] Many of the elements of the present invention, as it applies tothe treatment of atherosclerosis, are discussed in M. Strahilevitz,Lancet, Vol. 340, Jul. 25, 1992, p. 235, which is incorporated herein byreference.

[0025] One aspect of the present invention is to provide novelextracorporeal treatments for atherosclerosis based on specific affinityadsorption. The present invention also improves the efficacy ofextracorporeal LDL affinity adsorption by combining it with affinityadsorption of ligands other than LDL and other lipids, that are alsoetiological in atherosclerosis.

[0026] Another aspect of the present invention is providing means forreducing the level of oxidized LDL in the body, using as affinityadsorbents specific antibodies to oxidized LDL, or using as specificadsorbent enzymatic digestion fragments of such antibodies, or syntheticfragments of such antibodies.

[0027] Yet another aspect of the invention is improving theimmunoaffinity adsorption of LDL through the utilization of specificsynthetic fragments of antibody (G. W. Welling et al., Journal ofChromatography, Vol. 512, 1990, pp. 337-43), with synthetic fragmentsthat are specific to LDL. Yet another aspect is providing means forextracorporeal affinity adsorption of autoantibodies to oxidized LDL,which may be etiological in atherosclerosis, by using as the specificadsorbent oxidized LDL (the antigen) such as malondialdehyde LDL(Salonen, Lancet, supra), or to use as the adsorbent of oxidized LDLautoantibodies, Staphylococcal Protein A (Strahilevitz, Lancet, supra).Rather than Staphylococcal Protein A, a recombinant StaphylococcalProtein A or Staphylococcal Protein A component, or other syntheticpeptides of Staphylococcal Protein A may be utilized, as may Protein Gor its components. Bensinger, U.S. Pat. No. 4,614,513; R. Lindmark etal., J. Immunological Methods, Vol. 62, 1983, p. 1. As used herein,except when the context clearly indicates otherwise, the terms “ProteinA” and “Protein G” include all such variations.

[0028] When fragments of antibodies are used in the present invention asaffinity adsorbents, they can be produced by enzymatic (e.g., papain orpepsin) digestion of the intact antibody to produce Fab, (Fab′)2, or FVantigen-binding fragments, or they can be produced by other methodsknown to those skilled in the art for the synthesis of peptides, such assolid phase synthesis (R. A. Houghten, Proc. National Academy of ScienceUSA, Vol. 82, August 1985, pp. 5131-35; R. E. Bird et al., Science, Vol.242, 1988, pp. 423-42) or through genetic engineering in a suitablevector such as E. Coli or phage (J. W. Larrick, Pharmacological Reviews,Vol. 41(4), 1989, pp. 539-57). The use of fragments, rather than intactantibodies, as the affinity adsorbent may increase the adsorptioncapacity and reduces side effects that may be associated with the Fcnon-antigen binding part of the antibody molecule.

[0029] Another objective of the invention is to provide forextracorporeal chelation therapy for cancer, autoimmune diseases anddegenerative diseases, such as rheumatoid arthritis.

[0030] An additional objective is to provide extracorporeal combinedtreatment of cancer based on combining extracorporeal chelation andextracorporeal adsorption of enhancing tumor antibodies and theircomplexes by utilizing one or more of the following specific adsorbents,(a) Tumor specific antigen and (b) Staphylococcal Protein A or ProteinG.

BRIEF DESCRIPTION OF THE DRAWINGS

[0031]FIG. 1 is a diagrammatic view illustrating an affinity filtrationdevice utilized for extracorporeal chelation therapy in accordance withthe present invention.

[0032]FIG. 2 is a multi-hollow fiber dialyzer or diafilter utilized forextracorporeal chelation therapy in accordance with the presentinvention.

[0033]FIG. 3 is a detail of one hollow fiber of the device of FIG. 2.

[0034]FIG. 4 is a diagrammatic view of an extracorporeal affinityadsorption device for use with the present invention.

[0035]FIG. 5 is a diagrammatic view of two extracorporeal affinityadsorption devices connected in series for use in the present invention.

[0036]FIG. 6 is a diagrammatic view of two extracorporeal affinityadsorption devices connected in parallel for use in the presentinvention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The following are examples of the preferred embodiments ofdevices and methods of the present invention. All of the examplesutilize selective affinity binding of one ligand to another. The ligandwhich is held in an extracorporeal device will be referred to herein asa specific affinity “adsorbent,” even in cases in which that ligand isin solution or suspension, and the process of binding a chemical speciescarried in a fluid by means of the specific affinity adsorbent will bereferred to as “affinity adsorption.”

[0038] Affinity Filtration Chelation

[0039] Referring now to the drawings, and in particular to FIG. 1, anapparatus is provided which corresponds to the apparatus of my U.S. Pat.No. 4,375,414, but in which a chelating agent is utilized as thespecific affinity adsorbent. A column 1 is divided into a firstcompartment 2 and a second compartment 3, by a semipermeable membrane 4.Such membranes having various pore sizes and which are permeable tomolecules of molecular weight below a particular weight only (“cut off”)are available commercially. Preferred is a membrane with a pore size of0.001 micron to 0.01 micron, a suitable molecular weight cut off is1,000 to 10,000 daltons. One suitable membrane is a polysulphonemembrane (M. Amato et al., The International Journal of ArtificialOrgans, Vol. 11 (3), 1988, pp. 175-80. Another suitable membrane is madeof modified Cuprophan (Hemophan). (S. K. Mujais and P. Ivanovich in“Replacement of Renal Function by Dialysis”, Third Edition. KluwerAcademic Pub., J. F. Maher Editor, 1989, pp. 181-98.

[0040] The membrane is preferably pleated to increase its surface area.The membrane is mechanically supported by a rigid mesh screen 5, facingcompartment 3, thus avoiding contact of the mesh support material withthe blood flow. Positive pressure pump 6 and negative pressure pump 7are connected to compartments 2 and 3 and can be optionally operatedwhen increased pressure across the membrane is needed for enhancing themass transfer across the membrane between compartments 2 and 3. Theoverall surface area of the membrane is between 0.5 m²-3 m². The pumpsare connected to a control 14 to enable automatic operation. Compartment2 has an inlet 8 for a catheter 8 a, which is to be connected to a vein8 b of the patient (when vein to vein connection is used, which is thepreferred operation). Catheter 8 a may however be connected to an arteryof the patient when desired. Outlet 9 is connected to a catheter 9 a,which is to be connected to another vein 9 b of the patient. Togetherthey comprise a blood flow passage through compartment 2. The secondcompartment 3 includes the chelating agent. Preferably the chelatingagent is a multivalent chelating agent such as a biotechnology graderesin marketed by Biorad Corp. under the registered trademark Chelex100. This resin is a styrene divinylbenzene copolymer containing pairediminodiacetate ions, which act as chelating groups for bindingpolyvalent metal ions. Chelation is based on coordination bindingbetween the chelating agent and the heavy metals, which is similar to acovalent bond, but in which both electrons are donated by the same atom.Chelating binding differs from ion exchange by its high selectivity forheavy (transition) metal ions and by its much higher bond strength.Chelex 100 resin has the following selectivity for some cations (ahigher number indicates greater affinity): Hg⁺⁺=1060, Cu⁺⁺=126,Na⁺=1×10⁻⁷.

[0041] The Chelex 100 resin is obtained in 200 mesh size and is groundto particles with a diameter of 5 to 30 microns. The dialysis fluid is astandard renal dialysis fluid, preferably bicarbonate type buffered topH 7.4.

[0042] While Chelex 100 is preferred for some applications, otherchelating agents can be used when bound to macromolecular organic orinorganic particles, such as triaminepenta acetic acid or deferoxaminebound to a suitable matrix, such as silica. Other chelating agents arederivatives of iminodiacetic acid such as EDTA and matrix bound glycinehydroxamic acid.

[0043] Peristaltic pumps 10 and 11 are optional and may be used asneeded to accelerate blood flow into and out of compartment 2. The bloodflow through the device is in the range of 25 ml to 250 ml per minute.The system described thus far can be used without online regeneration,with the replacement of buffer and chelating agent as needed via inlet12 and outlet 13. This mode of treatment can be operated manually orautomatically, by the use of control 14 to operate the valving of inlet12 and outlet 13, in conjunction with reservoir 15 that contains freshChelex dialysis fluid suspension. Reservoir 16 collects “used up” Chelexwith its chelated heavy metals. Drain 17 can be used to clear reservoir16, for discarding, chemical analysis or off line regeneration of thechelating agent.

[0044] Optionally, the system can operate with either manual orautomatic on-line regeneration of the chelating agent. The automaticregeneration is identical to Strahilevitz, U.S. Pat. Nos. 4,375,414 and4,813,924, except for the use of a buffer pH 4 rather than pH 2.5.

[0045] Because the chelating agent specifically binds heavy metals suchas Cu⁺⁺ and Fe⁺⁺ that are mass transferred across the membrane barrier,a continuous gradient is present for the heavy metals that continue tomass transfer from compartment 2 to compartment 3 across membrane 4 bydiffusion and/or convection, as long as compartment 3 contains freechelating groups that are available to bind heavy metals. When pressurefiltration is utilized, by operating the optional positive pressure pump6 and/or negative pressure pump 7, the process of mass transfer isaccelerated by increased convection.

[0046] Optional means are provided for on-line continuous regenerationof the chelating agent. Preferably regeneration is achieved by use of aweak acid solution at pH 4 or higher. However, elution can also beachieved by use of a concentrated salt solution, or by the use of a lowmolecular weight free chelator, such as EDTA (specific elution). Whenon-line regeneration is used, the Chelex bound heavy metals buffersolution is transferred through outlet 25 to compartment 18 withoptional operation of peristaltic pump 27. The buffer is pressurefiltered through membrane 19 to compartment 20 (permeable to the bufferbut not to the Chelex). The positive pressure for filtration is providedby pump 28. The elution (regeneration) buffer is then transferred fromcompartment 21 to compartment 18 and again positive pressure pump 28transfers the heavy metals eluted from the Chelex to compartment 20 andthrough outlet 23 to compartment 24, from which it can be discarded orused for chemical analysis. Buffer pH 7.4 is added from reservoir 22 tocompartment 18 and the regenerated Chelex is returned to compartment 3via conduit 26. Optional peristaltic pump 29 is used to acceleratetransfer. Alternatively, other configurations can be used for on-lineregeneration, such as the one utilized in S. P. Halbert, U.S. Pat. No.4,637,880.

[0047] While the method described thus far uses Chelex with beaded,preferably regular spherical, form, other forms of matrix can be usedsuch as irregular beads or fibers, either natural or synthetic, to whichthe chelating moiety can be covalently bound or physically trapped(immobilized).

[0048] The chelating moiety can also be covalently bound tosemipermeable membrane 4, when the membrane is made of synthetic polymeror from natural or modified polymer. The binding can be to the membraneside facing the blood flow, the membrane side facing the dialysischelator flow, or to both sides. The physical configuration of thematrix is not limited to any particular form, as long as the matrixconfiguration and its particle size prevents the chelating agent frombeing substantially transferred from compartment 3 to compartment 2.

[0049] The affinity chelation filtration can be utilized for affinityadsorption of a plurality of ligands, for example when free antibodiesor antibodies bound covalently to a matrix or polymerized antibodies areincluded in compartment 3 and semipermeable membrane 4 is permeable tothe antigen or hapten to which the antibodies are specific. An examplefor such antibodies are the antibodies to free cholesterol.

[0050] Affinity Adsorption Chelation

[0051] The configuration and process of this treatment is similar toaffinity filtration chelation, except that the semipermeable membrane 4and its mesh screen membrane support are excluded from the device ofFIG. 1, along with pumps 6 and 7. A chelating agent, Chelex 100, groundto particle size of between 1-5 microns is encapsulated in a suitablemicrocapsule or macrocapsules. The macrocapsules used are those utilizedby A.M. Wallace and A. Wood, Clinical Chimica Acta, Vol. 140, 1984, pp.203-12, for encapsulating antibodies and have an average diameter of 30microns, or they are thermoplastic based macrocapsules (P. Aebischer etal., J. Biomech. Eng., Vol. 113 (2), 1991, pp. 178-83) having a diameterof 560±65 microns. Another suitable encapsulation of Chelex is bymodification of the method of L. Marcus et al., American Heart Journal,Vol. 110, No. 1, Part 1, July 1985, pp. 30-39. The method used by Marcuset al. involved encapsulation of anti-digoxin antibodies in 0.2 micronpolyacrolein microspheres, which are then encapsulated in 500-800 microncross-linked agarose macrospheres. For encapsulation of Chelex, theChelex is directly encapsulated in the cross-linked agarose macrospheresomitting the polyacrolein microencapsulation. Marcus et al. used theirencapsulated antidigoxin antibodies in a column for extracorporealadsorption treatment of digitalis intoxicated dogs. The same treatmentwas used in digitalis intoxicated humans. H. Savin et al., AmericanHeart Journal, Vol. 113(5), May 1987, pp. 1078-84.

[0052] When Chelex is used in free form, particles of about 30 to 300microns are preferred. Other matrixes and other chelating agents boundto the matrixes can be used. The matrixes can be of various chemicalcomposition including, for example: natural polymers such as celluloseand dextran; various synthetic polymers and copolymers such aspolyacrylamide, polystyrene, and polyvinyl polystyrene copolymer, andglass and silica. One suitable matrix is heparinized silicone describedin D. R. Bennett et al., U.S. Pat. No. 3,453,194. Various matrixes andmethods for their activation for covalent ligand binding are describedin P. D. G. Dean et al., Editors “Affinity Chromatography: A PracticalApproach,” IRL Press, Oxford 1985, pp. 1-73. The configuration of thematrix is also not limited to a particular form; examples of suitableforms are beads (in particular, spherical in shape), fibrous matrixes,macroporous matrixes, and membranes including hollow fibers.

[0053] One possible configuration of the device is that of a typicalmulti-hollow fiber dialyzer or diafilter design. Such a design consistsof a bundle of hollow fibers encased in a tubular housing.

[0054] In this configuration compartment 2 corresponds to the innerspace of the hollow fibers and compartment 3 to the outer space of thehollow fibers.

[0055] In FIG. 2, blood flows from patient's vein through inlet 34 tocompartment 32 (inner space of fibers). Heavy metals which pass acrosssemipermeable membrane 40 are bound by chelation to Chelex suspended incompartment 33. The blood that flows out through outlet 35 is connectedto another vein of the patient. This blood is relatively free from heavymetals. Inlet 36 is used to replace used up Chelex suspension with newChelex suspension. The old Chelex suspension is drained through drain41. This step is optional. Optionally also, Chelex may be regeneratedonline by the same online regeneration means described with respect toFIG. 1. “Used up” Chelex suspension is transferred from compartment 33through outlet 39 to the regeneration unit. Regenerated Chelexsuspension is returned to compartment 33 from the regeneration unitthrough inlet 38. Suitable pumps may be utilized if it is desirable toincrease the blood pressure in compartment 32 and across membrane 40,thus increasing the rate of mass transfer from compartment 32 tocompartment 33 by a filtration process.

[0056] The space between fibers is sealed by sealing resin 37.

[0057]FIG. 3 illustrates a single fiber in the unit. A typical fiber'smembrane thickness is 6-30 microns. The combined inner membrane surfacearea is typically 0.75 to 1.2 meter².

[0058] Commercially available dialysers that can be used are Freseniusmodel F60 or Asahi PAN 150.

[0059] Another embodiment of affinity adsorption device for use in thepresent invention is shown in FIG. 4. The device of FIG. 4 can be usedto treat either blood or plasma. Particularly suitable for direct bloodtreatments are devices in which the matrix-bound chelator isencapsulated or when the matrix is a spiral structure such as forexample natural polymer or synthetic polymer membrane to which thechelating moiety is covalently bound. When plasma is treated in thedevice, a plasma separator is first used to separate on-line thepatient's plasma from the cellular elements of blood. The physicalconfigurations may include beads, in particular spherical beads, fibers,macroporous matrixes, membranes, and hollow fibers.

[0060] Blood may be directly treated, preferably when the matrix boundchelator is encapsulated. If plasma is treated, then the patient's bloodflows via conduit 115 to plasma separator 103 (e.g., a centrifugalcontinuous plasma separator such as marketed by Cobe (Cobe IBM 2997) orpreferably a membrane filtration plasma separator such as Kaneka Sulfoxor Cobe TPE). The blood cells are returned to the patient via conduit124 and the plasma is passed through conduit 117, via inlet 105 tocolumn 106.

[0061] When encapsulation of matrix chelate is not utilized in thesystem and the method utilizes treatment of plasma, on-line manual orautomatic regeneration can be used using a modification of the methodsof Strahilevitz, U.S. Pat. No. 4,813,924 or the method of Halbert, U.S.Pat. No. 4,637,880.

[0062] In the on-line regeneration mode, with inlets 105 and 111 closed,outlet 112 to reservoir 136 closed, and either valve 119 or valve 139closed, valve 130 is opened and buffer pH 7.4 is transferred fromreservoir 125 to column 106. This is an optional step utilized when itis desirable to wash some of the patient's plasma that is present incolumn 106 into the patient's circulation (with valve 119 open) or todrain this washed volume of plasma through drain 141. This is done whenit is desirable to reduce the amount of plasma proteins that is exposedto the eluting buffer. A small volume of buffer pH 7.4 is used, in orderto minimize the volume of buffer introduced into the patient, when theoption of returning the plasma to the patient is used. Alternatively theremoval of plasma from column 106 back to the patient, can beaccelerated by using positive pressure filtration with operation of pump131.

[0063] In the elution step, with all valves except valves 129 and 139closed, eluting buffer pH 5 is transferred to column 106 from reservoir127; after equilibration, valve 129 is closed, valve 139 is opened andthe buffer, including the free heavy metal cations passes through drain141. Optionally, pressure filtration can be utilized with operation ofpump 131. Optional filter 134 is permeable to buffer, heavy metalcations, and plasma proteins about the size of LDL, but not to Chelexand plasma proteins larger than LDL; the drained fluid is then collectedin reservoir 135 and can be discarded or used for chemical analysis. Inthe next step column 106 is equilibrated with buffer pH 7.4, transferredfrom reservoir 125 through conduit 126.

[0064] When on-line regeneration is not used, replacement of usedadsorbent by fresh adsorbent can be done manually, or automatically byautomatic control of valves 137′ and 138 with addition of freshadsorbent from reservoir 137 and collecting used adsorbent in reservoir136.

[0065] Column 106 is then ready for re-use. Preferably vein to veincatheterisation is used, but when needed artery to vein catheterisationis utilized. When needed peristaltic pumps are used to accelerate fluidand mass transfer across the conduits 104, 105, 115, 117, 124 and 141.

[0066] The affinity adsorption method is well adapted to the concurrentadsorption of a plurality of ligands. On-line regeneration can be usedwhen needed, and is particularly simple when the regeneration of thevarious adsorption ligates can be regenerated by the same regenerationmeans, such as by an acidic pH buffer, for example. The variousadsorbents can be present in free form or can be encapsulated inmicrocapsules. Free form adsorbents are preferable because of theirmechanical strength and suitability for regeneration, when desired.Encapsulated adsorbents will generally not be suitable for regeneration.

[0067] In FIG. 4, column 106 contains a first adsorbent 107 and a secondadsorbent 108. Illustratively, the first adsorbent 107 is Chelex 100 inbead form, with a bead diameter in the range of 5 to 30 microns whenencapsulated and 30 to 300 microns when free. The bead is preferably infree form, but can be encapsulated as previously described. Themicrocapsule membrane, when present, is permeable to heavy metals butnot to Chelex or to plasma proteins. The Chelex specifically adsorbsheavy (transition) metals which catalyze oxidation of LDL. The secondadsorbent 108 is cyanogen bromide activated cross-linked agarose(Sepharose, Pharmacia Fine Chemicals), with a bead diameter in the rangeof 212-300 microns, prepared according to R. E. Ostlund, Jr., ArtificialOrgans, Vol. 11(5), 1987, pp. 366-74. The Sepharose is covalently boundto monoclonal antibodies to LDL. (R. L. Wingard et al., supra).According to Ostlund, supra, LDL is adsorbed by the antibodies. Aspreviously described, rather than intact antibodies, antibody fragmentscan be used. The combined effects of significant reductions of bothoxidant and LDL levels have a major impact on the atheroscleroticprocess.

[0068] Plasma which flows from column 106 though outlet 104 issubstantially free of the species sought to be removed. Drains 111 and112 can be used as needed for the removal of buffer and binding species,and for the addition of fresh binding species. Used binding species(e.g., anti-LDL antibodies and Chelex) can be regenerated off line, ifneeded. It should also be recognized that antibodies or fragments,“humanized” or hybrid antibodies (or fragments) can be used rather thanmouse antibodies. J. W. Larrick, supra. In synthesizing antibodyfragments, solid phase peptide synthesis methods (R. A. Houghten, supra)or genetic engineering methods (R. E. Bird et al., supra) can beutilized.

[0069] The advantage of Ab fragments over intact antibody is the reducedlikelihood of side effects of the immunoadsorption treatment,particularly when whole blood is used for adsorption and the antibody orfragment is not encapsulated thus enabling contact of the mouse antibodywith the patient's immune cells.

[0070] Additional adsorbents that can be utilized in the treatment ofatherosclerosis include oxidized LDL, which will adsorb autoantibodiesto oxidized LDL (cf. Salonen et al., Lancet, supra; Strahilevitz,Lancet, supra). Instead of the oxidized LDL, autoantibodies to oxidizedLDL and their complexes can be adsorbed by use of Sepharose 4BCL ProteinA, sold by Pharmacia Fine Chemicals. When Protein A is used as theadsorbent, the patient may need administration of replacement humangamma globulin. Additionally it may be desirable to adsorb oxidized LDLby using matrix bound antibodies to oxidized LDL as the adsorbent.

[0071] When affinity adsorption is used in accordance with the presentinvention for the adsorption of antigens or haptens, such as adsorptionof LDL or oxidized-LDL in the treatment of atherosclerosis or adsorptionof rheumatoid factor (autoantibodies to Human IgG) in the treatment ofrheumatoid arthritis, for example, the application of the analyticalmethod of J. Goding et al., J.Immunological Methods, Vol. 20, 1978, pp.241-53, to extracorporeal affinity adsorption in accordance with thepresent invention, is a general method for the removal of antigens orhaptens from the body. It should be clearly realized that any antigen orhapten can be removed from the body in accordance with this invention.In accordance with this method first Protein A or genetically engineeredProtein A peptide (R. Lindmark et al., supra) is covalently bound to anyof the matrixes described in the current invention, for exampleSepharose 4BCL. The antibody specific to the antigen, for examplemonoclonal antibody specific to LDL, is added to the Sepharose-boundProtein A. It binds to Protein A through its Fc part, and its Fabantigen binding part is available to bind the antigen (LDL). The matrixbound Protein A-LDL-antibody is incorporated in the extracorporealimmunoadsorption (affinity adsorption) treatment column as described inthe foregoing examples, for the treatment of atherosclerosis. Clearly itis possible to use Protein G instead of Protein A in this system.

[0072] When larger beads of cross linked Sepharose are used as matrix,they are prepared according to Ostlund, supra.

[0073] In the treatment of cancer the affinity adsorbents can includefor example: Chelex 100 to reduce oxidation and Staphylococcal ProteinA, or tumor specific antigens to remove enhancing tumor antibodies andtheir complexes.

[0074] An additional component of the combined treatment is toadminister a radioactive drug or conventional drug conjugated to anantibody specific to a tumor antigen (such as Adriamycin conjugated toan antibody to Human-Alpha-Fetoprotein, R. Yang et al., Antibody,Immunoconjugates and Radiopharmaceuticals, Vol. 5, 1992, pp. 201-07), inconjunction with adsorption of the antibody-drug conjugate from blood.K. Norrgren et al., Antibody Immunoconjugates and Radiopharmaceuticals,Vol. 4(4), 1991, pp. 907-14.

[0075] The utilization of tumor-targeted radiolabeled antibody inconjunction with immunoadsorption of the radiolabeled antibody from thecirculation to improve tumor imaging was reported by J. L. Lear et al.,Radiology, Vol. 179, 1991, pp 509-12. The adsorbent they used was anantibody to the radiolabeled anti-tumor antibody. The adsorbing antibodywas utilized in an extracorporeal column in which it was covalentlybound to a matrix. C. Hartmann et al., Journal of Pharmacokinetics andBiopharmaceutics, Vol. 19(4), 1991, pp. 385-403, evaluated the removalof radiolabeled antibody by extracorporeal adsorption, also usingantibody to the radiolabeled antibody as the adsorbent. They found thatthe method would be effective for enhancing tumor imaging and forincreasing the efficacy and reducing the toxicity of antibody-targetedanti-tumor drugs. These authors also cite two additional groupsreporting similar results.

[0076] In accordance with the present invention, the anti-tumor drug orradiolabeled antitumor antibody is adsorbed in an extracorporeal columnutilizing Staphylococcal Protein A as the adsorbent. This is a simplerand less expensive adsorbent and has the additional advantage ofadsorbing enhancing antibodies and immune complexes; this removal ofenhancing antibodies and immune complexes has an important therapeuticeffect on cancer. As previously mentioned, when Protein A is used as theaffinity adsorbent, it may be necessary to administer intravenously, tothe subject being treated, plasma or a plasma constituent such as gammaglobulin.

[0077] It should be clearly understood that in enhancing tumor imagingutilizing antibody-targeted radioactive ligand, as disclosed in Hartmannet al., supra, Lear et al., supra, or Norrgren et al., supra, Protein Aor Protein G can be utilized as the adsorbents.

[0078] Moreover, the radioactive imaging ligand may be incorporated in ahapten or antigen, preferably conjugated to the targeting antibody (orantibody fragment) by a spacer arm. The affinity adsorbent may then bean antibody to the free hapten or antigen, and all of the methodsdiscussed above for binding drugs bound to targeting antibodies may beutilized. Engineered targeting antibody fragments are disclosed in D. J.King et al., Antibody, Immunoconjugates, and Radiopharmaceuticals, Vol.5(2), 1992, pp. 159-70.

[0079] In the treatment of cancer the adsorption treatment will also becombined with conventional therapy such as chemotherapy.

[0080] Circulating immune complexes can also be adsorbed by C1qsubcomponent of complement bound to specific antibodies to C1q, whichare covalently bound to the matrix. T. Bratt and S. Ohlson, J. Clin.Lab. Immunol., Vol. 27, 1988, pp. 191-95. In combined treatment ofdegenerative diseases (such as rheumatoid arthritis, for example) theadsorbents include Chelex 100 and Staphylococcal Protein A, or matriximmobilized human IgG to bind the rheumatoid factor which is anautoantibody to IgG.

[0081] When whole blood is treated in the column, the optional plasmaseparation system is bypassed and the blood flows from vein 102Adirectly to column 106 via inlet 105. Optional membrane 113 and membrane114 are permeable to blood cells and plasma, but not to adsorbent-boundmatrix, which in this application when used in particle form utilizesparticles in the range of 300-800 microns in diameter to ensure freeflow of blood cells. The matrix can be in various other configurationssuch as fibers, membrane, capillaries, open porosity cavernous structureand the like. The matrix can be made of blood compatible syntheticpolymer, natural polymer and silica as examples. The filter 134 may bemade of smaller pore size when molecules smaller than LDL, such as freecholesterol, are to be removed. When LDL is removed, filter 134 ispermeable to molecules the size of LDL but not larger molecules.

[0082]FIG. 5 illustrates the use of two or more devices, eitherfiltration adsorption or direct adsorption when each of the specificadsorbents is contained in its own column. The devices and treatmentprocess can be operated manually or automatically. One or more of thedevices can be regenerated on line or off line. Either whole blood orplasma is adsorbed. Pumps as needed are included in the system tooptimize fluid flow through the system. Pumps are also utilized asneeded to increase trans-membrane pressure, when the filtrationadsorption process is used.

[0083] Referring to FIG. 5, a catheter 201 is inserted in vein 202 ofpatient 203, optionally passed through continuous plasma cell separator204 that is of either centrifugal or membrane type. The fluid (blood orplasma) is introduced into column 205 through inlet 206. Heavy metals inthe fluid are adsorbed to Chelex 100 beads 218. The fluid leaves column205 via outlet 207. It has a significantly reduced content of heavymetals such as Fe⁺⁺ and Cu⁺⁺. The fluid is then introduced to column 208via inlet 209. IgG and antibodies as well as antibody complexes areadsorbed on beads of Sepharose 4BCL/Protein A 210. Suitable filters arepositioned in the columns as described in reference to FIG. 4. The fluidleaving through outlet 211 has a reduced level of antibodies andcomplexes. The fluid is returned to the patient via catheter 212 andvein 217. When the plasma cell separator is in use the cellular elementsof the blood are returned to the patient via line 213, catheter 214 andvein 215.

[0084] The columns can be connected to the patient in parallel, ratherthan consecutively, as illustrated in FIG. 6.

[0085] With either the manual or automatic operation of valving, thepatient's blood or plasma can be transferred to column 301 and 302either consecutively, with valve 303 open when valve 304 is closed andvice versa, or concurrently with valves 303 and 304 both open at thesame time.

[0086] The method of Halbert, U.S. Pat. No. 4,637,880 may be used toregenerate one of two extracorporeal devices while the other devicecontinues to be used, without removing either device from the mammalbeing treated, using any of the devices of the present invention.

[0087] In the utilization of the methods of the invention, with orwithout the optional on-line regeneration step, heparin or anothersuitable anticoagulant may be administered intravenously or into thedevice as required, as is well known to those skilled in the art ofextracorporeal treatment. See for example, Bensinger, U.S. Pat. No.4,614,513.

[0088] Particularly when no regeneration of adsorbents is utilized,other columns can replace columns described in the current invention.For example, the column of Kuroda et al., U.S. Pat. No. 4,627,915 can beused to adsorb IgG and immune complexes, and the column of Ambrus etal., U.S. Pat. No. 4,612,122 can be used to remove heavy metals.

[0089] The present invention also includes the method of administering adrug bound (covalently or by other chemical binding) to an antibody suchas an antibody specific to a tumor or to a tissue-specific antigen.Administration of the drug-antibody moiety is followed by a step ofextracorporeally adsorbing the drug-hapten moiety by an antibodyspecific to the drug. The antibody in the extracorporeal device willthus adsorb both the drug-antibody moiety and the free drug in thecirculation of the patient. The extracorporeal adsorption is preferablybegun sufficiently long after the drug-antibody moiety is administeredto permit the drug to reach a target in the mammal, although in somecases concurrent initiation of administration and adsorption ispreferred. Generally, the time delay will typically be on the order ofseveral minutes to forty-eight hours. An example of the drug isAdriamycin bound to a targeting antibody; the antibody in theextracorporeal specific affinity device will then be an antibody toAdriamycin. An example of the tissue specific antigens is thyroid glandspecific antigen; an example of a tumor-specific antigen is humanalpha-fetoprotein. Both the targeting antibody, to which the drug isinitially bound, and the adsorbing antibody in the extracorporeal devicemay be an antibody fragment produced for example by synthesis or byenzymatic digestion treatment of a complete antibody. The adsorbentantibody is preferably linked to a matrix by a spacer arm of three tothirty carbon atoms; the targeting antibody is likewise preferablyattached to the drug by a spacer. The adsorbing antibody may be made asa mirror image antibody, which binds to a site on the drug differentfrom the site to which the targeting antibody is bound, by the methodset out in my U.S. Pat. No. 4,375,414. Use of antibody to the drug inthe extracorporeal device provides greater reduction in circulating drug(both bound and free), than does the antigen to which the targetingantibody is specific as used by Norrgren et al., sura.

[0090] Numerous other variations in the devices and methods of thepresent invention, within the scope of the appended claims, will occurto those skilled in the art in light of the foregoing disclosure.

I claim:
 1. A method for treating a disease state in a living mammal,the method comprising a step of administering to the mammal a moietycomprising a drug bound to an antibody, a step of drawing a fluidcontaining both the moiety and a chemical species which is etiologicalto the disease state or its symptoms from the mammal into anextracorporeal device, exposing the fluid to at least one affinityadsorbent for chemically binding the moiety and the chemical species inthe device, and a step of returning to the mammal at least a fraction ofthe fluid.
 2. The method of claim 1 wherein the antibody is a fragmentof a complete antibody.
 3. The method of claim 2 wherein the antibodyfragment is produced by synthesis.
 4. The method of claim 1 wherein theat least one affinity adsorbent comprises an antibody to the drug. 5.The method of claim 4 wherein the drug is Adriamycin.
 6. The method ofclaim 1 wherein the at least one affinity adsorbent comprises anantibody to the antibody constituent of the drug-antibody moiety.
 7. Themethod of claim 1 wherein the at least one affinity adsorbent comprisesan antigen specific to the antibody constituent of the drug-antibodymoiety.
 8. The method of claim 1 wherein the at least one affinityadsorbent comprises a species selected from the group consisting ofProtein A, Protein G, and C1q bound to anti-C1q antibody.
 9. The methodof claim 8 wherein the at least one affinity adsorbent is Protein A orProtein G.
 10. The method of claim 1 wherein the drug is radioactive.11. The method of claim 1 including a further step of administering tothe living mammal intravenously at least one plasma component.
 12. Themethod of claim 11 wherein the plasma component is gamma globulin.
 13. Amethod for diagnosing a disease state in a living mammal, the methodcomprising a step of administering to the mammal a moiety comprising aradioactive species bound to an antibody, a step of drawing a fluidcontaining both the moiety and a chemical species which is etiologicalto the disease state or its symptoms from the mammal into anextracorporeal device, exposing the fluid to at least one affinityadsorbent for chemically binding the moiety and the chemical species inthe device, and a step of returning to the mammal at least a fraction ofthe fluid.
 14. The method of claim 13 wherein the at least one affinityadsorbent comprises a species selected from the group consisting ofProtein A, Protein G, and C1q bound to anti-C1q antibody.
 15. The methodof claim 14 wherein the at least one affinity adsorbent is Protein A orProtein G.
 16. The method of claim 13 including a further step ofadministering to the living mammal intravenously at least one plasmacomponent.
 17. The method of claim 16 wherein the plasma component isgamma globulin.
 18. A method for treating a disease state in a livingmammal, the method comprising a step of administering to the mammal amoiety comprising a drug bound to an antibody, the drug being selectedfrom the group consisting of haptens and antigens, a step of drawing afluid containing the moiety from the mammal into an extracorporealdevice, exposing the fluid to an affinity adsorbent selected from thegroup consisting of Protein A and Protein G for chemically binding themoiety in the device, and a step of returning to the mammal at least afraction of the fluid.
 19. A method for diagnosing a disease state in aliving mammal, the method comprising a step of administering to themammal a moiety comprising a radioactive species bound to an antibody,the radioactive species being selected from the group consisting ofradioactive haptens and radioactive antigens, a step of drawing a fluidcontaining the moiety from the mammal into an extracorporeal device,exposing the fluid to an affinity adsorbent selected from the groupconsisting of Protein A and Protein G for chemically binding the moietyin the device, and a step of returning to the mammal at least a fractionof the fluid.